The present invention, in some embodiments thereof, relates to methods of generating corneal cells from pluripotent stem cells and cell populations comprising same.
The cornea is a unique, transparent structure that covers the iris, pupil, and anterior chamber, providing most of the eye's optical power. Together with the lens, the cornea refracts light and, as a result, aids in focusing. The cornea contributes more to the total refraction than the lens does, but, whereas the curvature of the lens can be adjusted to “tune” focus, the curvature of the cornea is fixed. The cornea has no blood vessels, its nourishment is obtained via diffusion from the tear fluid, the aqueous humor, and neurotrophins supplied by nerve fibers that innervate it. Thus, for example, disturbances in circulation of these fluids or inflammatory processes play a large role in the pathogenesis of corneal abnormalities.
The cornea is composed mostly of dense connective tissue. However, the collagen fibers are arranged in a parallel pattern, allowing light waves to constructively interfere, thus letting light pass through relatively uninhibited.
The corneal tissue is arranged in five basic layers: epithelium, Bowman's layer, stroma, Descemet's membrane and endothelium, each having a separate function. The epithelium is the outermost layer of the cornea, comprising about 10% of the tissue's thickness. The epithelium functions primarily to: (1) block passage of foreign materials, such as dust, water, and bacteria, into the eye and other layers of the cornea; and (2) provide a smooth surface that absorbs oxygen and cell nutrients from tears, then distributes these nutrients to the rest of the cornea. The corneal epithelium is maintained by stem cells (SCs) located at the periphery of the cornea, in a region known as the limbus. The corneal epithelium itself is devoid of its own stem cells. Limbal fibroblasts form the major cellular component of the limbal stroma upon which the LSCs reside, and they produce specific cytokines that promote corneal epithelial wound healing by the LSCs.
The changes associated with aging of the cornea include increased opacity, increased anterior surface curvatures, and possibly changes in refractive index distribution. Various refractive eye surgery techniques change the shape of the cornea in order to reduce the need for corrective lenses or otherwise improve the refractive state of the eye. In many techniques, reshaping of the cornea is performed by photoablation using an eximer laser.
If the corneal stroma develops visually significant opacity, irregularity, or edema, a cadaveric donor cornea can be transplanted.
Limbal auto-grafts have been applied to patients with a relatively high degree of success. In severe cases such as total limbal stem cell deficiency, allo-grafts may be obtained from patient's relatives or from post mortem donors. However, limbal tissue is highly immunogenic and the rate of graft rejection exceeds 35%, 5 years post transplantation. This method is further limited due to a shortage of donors. Further, living donors are at risk of developing limbal stem cell deficiency. To reduce this risk, a smaller limbal tissue may be grafted from donor and expanded ex vivo prior to transplantation. This technology does not allow treating patient immediately following injury, since it requires the cultivation of the cells for several weeks prior to grafting. Moreover, allogenic limbal stem cell transplantations to a patient are eventually rejected.
Synthetic corneas also exist (keratoprotheses), however, these are typically plastic inserts or may be composed of biocompatible synthetic materials that encourage tissue in-growth into the synthetic cornea, thereby promoting biointegration. Alternatively, orthokeratology offers the use of specialized hard or rigid gas-permeable contact lenses to transiently reshape the cornea in order to improve the refractive state of the eye or reduce the need for eyeglasses and contact lenses.
Homma et al. [Invest Ophthalmol Vis Sci. 2004 December; 45(12):4320-6] teach corneal epithelial differentiation of murine embryonic bodies on collagen IV-coated dishes.
Sajjad et al. [Stem Cells. 2007 May; 25(5):1145-55] teach generation of corneal epithelial-like cells by seeding human embryonic stem cells on collagen IV-coated dishes in the presence of medium that was conditioned by limbal fibroblasts. The resulting corneal epithelial cells were contaminated with skin cells.